Biochem. J. (1975) 148, 329-333 Printed in Great Britain

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Synthesis and Sidedness of Membrane-Bound Respiratory Nitrate Reductase (EC 1.7.99.4) in Escherichia coli Lacking Cytochromes By M. B. KEMP, B. A. HADDOCK and P. B. GARLAND Department oJ Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, U.K.

(Received 30 December 1974) The synthesis of nitrate reductase and its incorporation into the cytoplasmic membrane of Escherichia coli strain A1004a (5-aminolaevulinic acid auxotroph) does not require synthesis of cytochrome b. The synthesis of the apoprotein(s) of the cytochrome b of the respiratory pathway from NADH to nitrate appears to be inhibited by the absence of haem. No member of the respiratory pathway from NADH to oxygen is capable of reducing nitrate reductase directly. The site on nitrate reductase that oxidizes FMNH2 is located on the cytoplasmic aspect of the cytoplasmic membrane. Chang & Lascelles (1963) showed that a haemrequiring mutant of Staphylococcus aureus synthesized nitrate reductase in the absence of haem, and that incubation of broken-cell preparations with haem reconstituted electron transport from substrate dehydrogenase to nitrate. Somewhat similar findings for the NADH oxidase activity of a 5-aminolaevulinic acid-requiring auxotroph of Escherichia coli were reported by Haddock & Schairer (1973), who made the further discovery that reconstitution of haem and membrane-bound cytochrome apoprotein(s) was ATP-dependent (Haddock, 1973). In contrast with the findings of Chang & Lascelles (1963), De Groot & Stouthamer (1970) claimed that the synthesis of nitrate reductase activity was dependent on cytochrome synthesis in a 5-aminolaevulinic acid-requiring auxotroph of Proteus mirabilis. We have therefore explored the effects of inhibited cytochrome synthesis on nitrate reductase synthesis in a 5-aminolaevulinic acid-requiring auxotroph of E. coli. Experiments described in the present paper show that nitrate reductase synthesis by E. coli could be de-repressed even in the absence of cytochrome synthesis, and that the enzyme was then functionally isolatedfrom otherrespiratoryenzymes. Nevertheless, non-physiQlogical reductants such as FMNH2 or reduced Benzyl Viologen were still used by the nitrate reductase. Reduced Benzyl Viologen can probably permeate into cells of E. coli whereas FMNH2, if it resembles FMN, cannot (Wilson & Pardee, 1962). Thus the accessibility of nitrate reductase to these reductants in preparations of intact and disrupted cells, and spheroplasts, can indicate whether the site on the enzyme that reacts with FMNH2 is on the outer or inner surface of the cytoplasmic membrane. The interpretation is not complicated by the possibility that the reductants Vol. 148

reduce nitrate reductase indirectly through some other point or points in a complex respiratory chain. Materials and Methods Organism E. coli A1004a (Haddock, 1973) was derived from strain 201 (Haddock & Schairer, 1973) and generously given by Dr. H. U. Schairer, Max Planck Institut fur Biologie, 73-Tubingen, West Germany. Cell growth

Glycerol (1OOg/litre) was added to a broth culture, which was then divided into small (2ml) portions and stored at -15°C. A portion was thawed and added to broth (20ml) composed of Oxoid nutrient broth no. 2 (25 g/litre; Oxoid Ltd., London SEI 9HF, U.K.), supplemented with glucose (5g/litre) and 5OmM-KH2PO4-Na2HPO4, pH6.8. The inoculated broth was incubated anaerobically for 24-30h at 37°C. The cells were then harvested aseptically at room temperature by centrifugation and resuspended in 500ml of Hutner's minimal medium (Ornston & Stanier, 1966) containing (NH4)2SO4 (1 g/litre) to which the following additions (final concentrations shown) had been made after independent autoclaving: 50mM-KH2PO4-Na2HPO4, pH 8.0, glucose (5 g/litre), casamino acids (1 g/litre; Difco Laboratories, Detroit 1, Mich., U.S.A.), an amino acid mixture (methionine, 20mg/litre, isoleucine, 20mg/litre; valine, 50mg/litre) and potassium selenite (1 M). This large-scale culture was incubated anaerobically at 33°C for 15h, and then diluted 4- or 5-fold into fresh medium containing NaNO3 (lOg/litre) and 5-aminolaevulinic acid (10mg/litre; filter-sterilized), as appropriate, and incubated further as described in the Results section. Small-scale cultures were incubated anaerobically

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M. B. KEMP, B. A. HADDOCK AND P. B. GARLAND

in an evacuated vacuum desiccator. Large-scale cultures were flushed before incubation with O2-free N2 ('White spot' N2, 99.9% pure; BOC, London S.W.19, U.K.) and stirred with a magnetic bar. The cultures were checked for revertants by examining for growth after plating out on 0.5 % (w/v) glycerolagar medium with and without 5-aminolaevulinic acid. Cell harvesting and breakage Large-scale cultures were harvested by centrifugation at room temperature and washed thrice with 50mM-KH2PO4-Na2HPO4, pH6.8. The washed pellets were stored overnight at -15°C, resuspended in 50mM-KH2PO4-Na2HPO4, pH6.8 (5ml per g wet weight), and exposed to the output of a 100W ultrasonic probe (MSE Ltd., Crawley, Sussex, U.K.) until the turbidity, measured as extinction at 600nm, had decreased by at least 90%. The broken-cell suspension was used for enzyme assays without centrifugation. Enzyme assays All assays were made spectrophotometrically in a final volume of 2.5-3.0ml at 30°C in 50mM-KH2PO4Na2HPO4, pH6.8, in cuvettes of 1cm light-path. NADH oxidase was assayed at 340nm at an initial NADH concentration of 0.15mm. NADH-nitrate reductase was also assayed at 340rnm, but anoxically and with initiation of the reaction by addition of KNO3 to a final concentration of 10mM. Formate dehydrogenase (EC 1.2.2.1) was assayed at 600nm by using lOmm-sodium formate, 0.4mM-N-methylphenazoniummethosulphate and 50 uM-2,6-dichlorophenol-indophenol (Ruiz-Herrera et al., 1969). Nitrate reductase was assayed anoxically with reduced Benzyl Viologen as reductant at 600nm, or with FMNH2 at 450nm. In either case the cuvette was set up anoxically with the buffer and oxidized Benzyl Viologen (0.3 M) or0.2mM-FMN, which was then partially reduced by adding 5-201 of 25mMsodium dithionite in 10mM-NaOH until the extinction at 600nm had risen to about 1.0, or at 450nm had fallen to about 0.7. The preparation under assay was then added to a final concn. of 10100ug/ml. The reaction was initiated by adding KNO3 to a final concn. of 10mM. 8-Galactosidase (EC 3.2.1.23) was assayed at 420nm by using 0.8mM-o-nitrophenyl fJD-galactoside as substrate (Rickenberg et al., 1956). NADH-ferricyanide reductase (EC 1.6.99.3) was assayed at 420 rm with 0.2mM-NADH and 1.0mMpotassium ferricyanide in the presence of 1.0mMKCN. Cytochrome b was assayed in a wavelengthscanning spectrophotometer (Haddock & Garland, 1971), and protein by the method of Lowry et al. (1951) with bovine plasma albumin as the standard.

Reagents NADH was the disodium salt, grade II, from Boehringer Corp. Ltd., Uxbridge Road, London W5 2TZ, U.K. 2,6-Dichlorophenol-indophenol (sodium salt, grade I), N-methylphenazonium methosulphate (phenazine methosulphate), D-chloramphenicol, 5aminolaevulinic acid hydrochloride, lysozyme chloride (grade VI: EC 3.2.1.17), FMN (sodium salt) and o-nitrophenyl fl-D-galactoside were purchased from Signa Chemical Co. Ltd., Norbiton Station Yard, Kingston-upon-Thames KT2 7BH, Surrey, U.K. Benzyl Viologen and bovine albumin fraction V came from BDH Chemicals Ltd., Poole, Dorset BH12 4NN, U.K. Preparation of spheroplasts A slightly modified method of Kaback (1971) was used.

Results and Discussion Growth of the haem-deficient mutant In the presence of 5-aminolaevulinic acid the rate and extent of growth of the mutant AlOO4a was the same as that of the parent strain. In the absence of 5-aminolaevulinic acid the mutant grew more slowly than the parent strain even under anaerobic glucosefermenting conditions (see the Materials and Methods section). Nitrate reductase activity in the haem-deficient mutant Part of a large-scale culture of mutant A1004a which had grown overnight in the absence of nitrate was harvested. The remainder was divided into two parts which were diluted fivefold into fresh medium containing nitrate and incubated respectively with and without addition of 5-aminolaevulinic acid. Nitrate reductase activity was de-repressed in both cultures (Table 1, cultures 2 and 3 compared with culture 1), although the specific activity found in cells of the 5-aminolaevulinic acid-supplemented culture was generally between two and three times that of the unsupplemented culture. NADH oxidase and NADH nitrate reductase activities were very low in the cells grown in the absence of 5-aminolaevulinic acid, as was formate dehydrogenase. Restoration of NADH-nitrate reductase activity Haddock & Schairer (1973) and Haddock (1973) showed for E. coli strain A1004a that even in the absence of haem the apoprotein(s) of the cytochrome b of the respiratory pathway from NADH to oxygen was synthesized, associated with the cell membrane, and able to reconstitute with haem (if ATP was also present) to form a functionally active cytochrome. Experiments to determine whether the cytochrome b of the respiratory pathway from NADH to nitrate (Ruiz-Herrera & De Moss, 1969) behaved similarly were carried out as described in Table 1. It is clear 1975

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CYTOCHROMES AND NITRATE REDUCTASE IN E. COLI

Table 1. Effects of5-aminolaevulinic acid duringgrowth on the activities of respiratory enzymes in E. coli strain Al 004a, and on the restoration ofactivities in the presence or absence ofchloramphenicol An overnight culture anoxically grown in minimal medium without nitrate or 5-aminolaevulinic acid (see the Materials and Methods section) was divided into three equal parts. One part, culture 1, was harvested and assayed for the activities listed below. The other two parts were each diluted fivefold with fresh medium and incubated either for 5h with both nitrate and 5-aminolaevulinic acid, giving culture 2, or for 7h with nitrate, giving culture 3. All of culture 2 and half of culture 3 were harvested for assays, and these formed the basis for the experiment comparing the effects of growth conditions. An experiment to study the restoration of deficient respiratory enzyme activities was carried out as follows. The remainder of culture 3 was diluted with an equal volume of medium containing nitrate, and divided into three equal parts which were incubated for a further 3.5h respectively without 5-aminolaevulinic acid (culture 4), with 5-aminolaevulinic acid (culture 5) and with D-chloramphenicol (50mg/litre) and 5-aminolaevulinic acid (culture 6). All cultures were grown anoxically, harvested and assayed as described in the Materials and Methods section. Growth was measured as wet weight of the harvested cells from a given volume of culture; for cultures 2 and 3 it is expressed relative to culture 1 and for cultures 4-6 relative to culture 3, after allowing for dilution. Enzyme activities are expressed as ,umol of NADH or formate oxidized, or nitrate reduced. The absolute activities varied between experiments, but their large change in response to additions to the growth medium were consistent within any one experiment. The results refer to a single experiment, with results from another experiment given in parentheses to indicate variation of absolute activities between experiments. Cyto- Enzyme activities (&mol/min per mg of protein) No. of culture I 2 3 4 5 6

Additions to minimal growth medium None

NO3-, 5-aminolaevulinic acid

NO3-

NO3NO3-, 5-aminolaevulinic acid

N03-, 5-aminolaevulinic acid, chloramphenicol

chromeb (nmol/mg of Growth (fold) protein)